AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity

Li, Feixuan1; Liu, Xiao-Yu3; Ni, Lei2; Jin, Fan2

2022-12-01

10.1128/spectrum.03783-22

Microbiology Spectrum   影响因子和分区

2165-0497

["Transcriptional factors (TFs) and their regulons make up the gene regulatory networks. Here, we developed a method based on TF-directed activation-induced cytidine deaminase (AID) mutagenesis in combination with genome sequencing, called AIDmut-Seq, to detect TF targets on the genome. AIDmut-Seq involves only three simple steps, including the expression of the AID-TF fusion protein, whole-genome sequencing, and single nucleotide polymorphism (SNP) profiling, making it easy for junior and interdisciplinary researchers to use. Using AIDmut-Seq for the major quorum sensing regulator LasR in Pseudomonas aeruginosa, we confirmed that a few TF-guided C-T (or G-A) conversions occurred near their binding boxes on the genome, and a number of previously characterized and uncharacterized LasR-binding sites were detected. Further verification of AIDmut-Seq using various transcriptional regulators demonstrated its high efficiency for most transcriptional activators (FleQ, ErdR, GacA, ExsA). We confirmed the binding of LasR, FleQ, and ErdR to 100%, 50%, and 86% of their newly identified promoters by using in vitro protein-DNA binding assay. And real-time RT-PCR data validated the intracellular activity of these TFs to regulate the transcription of those newly found target promoters. However, AIDmut-Seq exhibited low efficiency for some small transcriptional repressors such as RsaL and AmrZ, with possible reasons involving fusion-induced TF dysfunction as well as low transcription rates of target promoters. Although there are false-positive and false-negative results in the AIDmut-Seq data, preliminary results have demonstrated the value of AIDmut-Seq to act as a complementary tool for existing methods.IMPORTANCE Protein-DNA interactions (PDI) play a central role in gene regulatory networks (GRNs). However, current techniques for studying genome-wide PDI usually involve complex experimental procedures, which prevent their broad use by scientific researchers. In this study, we provide a in vivo method called AIDmut-Seq. AIDmut-Seq involves only three simple steps that are easy to operate for researchers with basic skills in molecular biology. The efficiency of AIDmut-Seq was tested and confirmed using multiple transcription factors in Pseudomonas aeruginosa. Although there are still some defects regarding false-positive and false-negative results, AIDmut-Seq will be a good choice in the early stage of PDI study.","Protein-DNA interactions (PDI) play a central role in gene regulatory networks (GRNs). However, current techniques for studying genome-wide PDI usually involve complex experimental procedures, which prevent their broad use by scientific researchers."]

activation induced cytidine deaminase Pseudomonas aeruginosa protein-DNA interactions

SCI

英语

其他

[2020YFA0906900] ; [2018YFA0902700] ; [YJKYYQ20200033]

Microbiology

Microbiology

WOS:000898792300001

AMER SOC MICROBIOLOGY

Web of Science

1.Univ Sci & Technol China, Hefei Natl Res Ctr Phys Sci Microscale, Dept Polymer Sci & Engn, Hefei, Peoples R China
2.Chinese Acad Sci, Shenzhen Inst Synthet Biol, Shenzhen Inst Adv Technol, CAS Key Lab Quantitat Engn Biol, Shenzhen, Peoples R China
3.Southern Univ Sci & Technol, Sch Med, Shenzhen, Peoples R China

Li, Feixuan,Liu, Xiao-Yu,Ni, Lei,et al. AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity[J]. Microbiology Spectrum,2022,11(1).

Li, Feixuan,Liu, Xiao-Yu,Ni, Lei,&Jin, Fan.(2022).AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity.Microbiology Spectrum,11(1).

Li, Feixuan,et al."AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity".Microbiology Spectrum 11.1(2022).